One of the greatest challenges to modern biomedical science is the ability to measure minute amounts of specific molecules in samples with limited availability. U.S. Pat. No. 4,857,454 teaches an assay method using an antibody/analyte/second antibody-enzyme conjugate microparticle system. The method requires washing the microparticles to remove non-captured materials, a step that introduces a great deal of variability. In addition, the color generated in the microparticles diffuses into the solution surrounding the microparticles, which dilutes the signal representing the quantity of analyte. U.S. Pat. Nos. 5,674,699 and 7,851,229 disclose a method for assaying Hemoglobin Alc in a blood sample using a two-phase optical assay. The prior art of the two-phase assay only discloses a single functional component on the microparticle, a ligand to capture the analyte. The analyte Hemoglobin Alc in the blood sample is intensely colored and abundant enough in the erythrocytes (about 2 μM even in non-diabetics) so that it is feasible to use the method disclosed therein for measuring Hemoglobin Alc. The methods disclosed in those two patents however cannot perform an assay for an analyte that has insufficient or no inherent optical contrast. For example, cytochrome C is colored but the levels in cells are too low to be measured by using the methods disclosed therein.
Therefore, a previously unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with measuring analytes with no intrinsic optical properties.